JP7466876B2 - Self-assembling peptides - Google Patents

Description

The present invention relates to a self-assembling peptide, a sol-gel transition agent, a sol-gel transition composition containing the sol-gel transition agent as an active ingredient, and a sol-gel transition method thereof.

Among the peptides that form intermolecular interactions, there are some that form supramolecular fibers in water to give hydrogels (Non-Patent Documents 1-2). These hydrogels are being applied as biomaterials due to the advantages of peptides in general, such as biocompatibility and biodegradability. Examples of applications of the hydrogel include cell scaffolding materials (Non-Patent Document 3) and transport materials for pharmaceutical preparations (Non-Patent Document 4). Animal-derived gelling agents such as collagen and Matrigel are typically used for hydrogels. However, animal-derived gelling agents have quality differences in molecular weight distribution and component ratios between production lots. In addition, contamination from various organisms can have a significant effect on biological samples such as cells. As a result, the risk of allergies and unknown infectious diseases cannot be avoided, which has been problematic in terms of clinical application (Non-Patent Documents 5-9).

In recent years, synthetic low molecular weight peptide gelators have been attracting attention as a material that can overcome these problems. Synthetic low molecular weight peptide gelators began with EAK16 and RADA16, discovered by Shuguang Zhang et al. in 1993, and research and development has been ongoing to this day (Non-Patent Documents 10-12). These are produced by synthesizing high-purity peptides consisting of specific amino acid sequences using solution synthesis or solid-phase synthesis, and have the advantage that the quality of each production lot is stable and there is very little contamination.

Representative examples of low molecular weight peptide gelators include (A) amphipathic peptides in which one or two amino acids are linked to an aromatic moiety (such as a cinnamoyl group or an Fmoc group), (B) amphipathic peptides in which a long alkyl chain (such as a palmitoyl group) is linked to one end of a β-sheet-forming peptide, and (C) amphipathic peptides in which hydrophilic and hydrophobic amino acids are linked alternately (Non-Patent Documents 1-2). The fact that (A) and (B) contain a non-natural backbone can be a problem in biological or clinical applications. On the other hand, (C) is made only from natural amino acids and is therefore considered to have an advantage over (A) and (B).

Stimuli-responsiveness is also an important factor in expanding the applicability of peptide gels. Materials that show gel-to-sol or sol-to-gel transitions in response to external stimuli such as pH and temperature can switch cell scaffolding properties in vivo and control the release of substances contained in the gel, thereby increasing the added value of the material. Many examples of imparting stimuli-responsiveness using non-natural backbones have been reported (Non-Patent Documents 13-14). On the other hand, there are very few examples of stimuli-responsiveness in peptide gels composed only of natural amino acids. For example, Shuguang Zhang et al. (Non-Patent Document 15) reported that DAR16-IV, which is composed of alanine, aspartic acid, and arginine residues, among RADA16 analogs, shows a structural transition from β-sheet to α-helix with increasing temperature. On the other hand, it is known that RAD16-IV, which has a similar sequence, does not show a structural transition due to temperature change (Non-Patent Documents 10-12). However, a general design method that can impart stimuli-responsiveness to peptide gelators composed only of natural amino acids has not yet been found.

Dasgupta, A. et al., RSC Advances, 2013, 3, 9117-9149. Jonker, A.M. et al., Chemistry of Materials, 2012, 24, 759-773. Wu, E.C. et al., Advanced Functional Materials, 2012, 22, 456-468. Vermonden, T. et al., Chemical Reviews, 2012, 112, 2853-2888. Vukicevic, S. et al., Experimental Cell Research, 1992, 202, 1-8. Vallier, L. et al., Journal of Cell Science, 2005, 118, 4495-4509. Cooperman, L. and Michaeli, D., Journal of the American Academy of Dermatology, 1984, 10, 638-646. Lynn, A.K. et al., Journal of Biomedical Materials Research Part B Applied Biomaterials, 2004, 71, 343-354. Scott, M.R. et al., Proceedings of the National Academy of Sciences of the United States of America, 1999, 96, 15137-15142. Zhang, S. et al., Proceedings of the National Academy of Sciences of the United States of America, 1993, 90, 3334-3338. Zhang, S. et al., Biopolymers, 1994, 34, 663-672. Zhang, S. et al., Biomaterials 1995, 16, 1385-1393. Kopecek, J. and Yang, J., Angewandte Chemie International Edition, 2012, 51, 7396-7417. He, X. et al., Chemistry An Asian Journal, 2016, 11, 437-447. Zhang, S. and Rich, A., Proceedings of the National Academy of Sciences of the United States of America, 1997, 94, 23-28.

The object of the present invention is to develop and provide a self-assembling peptide that is biocompatible and capable of controlling the temperature of the sol-gel transition, which enables switching of cell scaffolding properties and release of substances contained in the gel, and to provide a method for causing the sol-gel transition of the self-assembling peptide at a desired temperature.

In order to solve the above problems, the present inventors focused on (Arg-Ala-Asp-Ala) _tetrapeptide (SEQ ID NO: 1) (JP Patent Publication No. 2011-46741), which is known as a gelling peptide composed of natural amino acids. The "(Arg-Ala-Asp-Ala) _tetrapeptide " is a polypeptide in which four peptide units consisting of the four amino acids Arg-Ala-Asp-Ala (SEQ ID NO: 2) are linked by peptide bonds. In this specification, amino acid residues are often represented by one letter. For example, an Arg (arginine) residue is represented by R, an Ala (alanine) residue is represented by A, an Asp (aspartic acid) residue is represented by D, a Gly (glycine) residue is represented by G, and a Pro (proline) residue is represented by P. Therefore, "Arg-Ala-Asp-Ala" is often represented as "RADA". The same applies to other peptides.

As a result of extensive research, the present inventors have found that an amphipathic mutant gelling peptide in which one of the two alanine residues in the C-terminal unit of (RADA) ₄ peptide is replaced with a glycine residue, and an amphipathic mutant gelling peptide in which the N-terminal alanine residue of the two alanine residues in the N-terminal unit of (RADA) ₄ peptide is replaced with a glycine residue, have the property of forming a sol at a lower temperature than (RADA) ₄ peptide. The present invention is based on the above new findings and provides the following.

(1) Any one of self-assembling peptides comprising m RADA and n RXDA (SEQ ID NO: 3) or RADX (SEQ ID NO: 4) arranged in any order, wherein X is G or P, 3≦m≦6, 1≦n≦2 and 2n<m, and the C-terminus of the self-assembling peptide is RXDA or RADX, or the N-terminus is RXDA.
(2) The self-assembling peptide according to (1), wherein X is G.
(3) A fusion peptide comprising the self-assembling peptide according to (1) or (2) and a functional peptide linked thereto via a covalent bond or supramolecular interaction.
(4) The fusion peptide according to (3), wherein the functional peptide is any one of functional peptides selected from laminin, VEGF, and N-cadherin.
(5) A sol-gel transition agent comprising the self-assembling peptide according to (1) or (2), or the fusion peptide according to (3) or (4).
(6) A sol-gel transition composition comprising the sol-gel transition agent according to (5) as an active ingredient.
(7) The sol-gel transition composition according to (6), comprising two or more types of sol-gel transition agents.
(8) The sol-gel transition composition according to (6) or (7), further comprising a self-assembling peptide consisting of (RADA) _P (p is an integer of 1 to 6) and/or a fusion peptide in which the functional peptide is linked to the self-assembling peptide via a covalent bond or supramolecular interaction.
(9) A cell scaffold material comprising the sol-gel transition agent according to (5) in a gel state, and/or the sol-gel transition composition according to any one of (6) to (8).
(10) A sol-gel transition method for the sol-gel transition agent according to (5) or the sol-gel transition composition according to any one of (6) to (8), which is in a sol or gel state, comprising a temperature treatment step of treating the sol-gel transition agent or the sol-gel transition composition at a temperature equal to or higher than its solization temperature or at a temperature equal to or lower than its gelation temperature.
(11) The method according to (10), wherein the concentration of the sol-gel transition agent and/or the sol-gel transition composition is 0.4% by weight or more and 10% by weight or less.
(12) The method according to (10) or (11), wherein the sol-gel transition agent or the sol-gel transition composition in a sol or gel state has a pH of 1 to 9.
(13) The method according to any one of (10) to (12), wherein the solation temperature or gelation temperature is within the range of 20 to 80° C.
This specification includes the disclosures of Japanese Patent Application No. 2019-028845, which is the priority basis of this application.

The self-assembling peptide of the present invention can provide a peptide that is biocompatible and capable of controlling the temperature of the sol-gel transition.
According to the sol-gel transition method of the present invention, the self-assembling peptide can be subjected to the sol-gel transition at a desired temperature.

This is a diagram showing a method for quantifying the solization of self-assembling peptides. When various peptide gels were prepared at the bottom of a 2 mL glass screw vial that was placed vertically, the distance from the bottom of the vial to the top of the gel sample was taken as the reference distance _h0 . Then, at a specified temperature, the vial was tilted 90 degrees, i.e., horizontally, and the distance from the bottom of the vial to the right end of the sample that had moved due to solization was taken as the movement distance h. The sol-gel state of each peptide sample was judged to be in a sol state if the h/ _h0 value was 1.3 or more, and in a gel state if the h/ _h0 value was less than 1.3. 1 shows the h/h ₀ value of each peptide sample at 20° C. The dashed line indicates the cutoff value (h/h ₀ =1.3) for determining the sol-gel state. 1 shows the h/h ₀ value of each peptide sample at 80° C. The dashed line indicates the cutoff value (h/h ₀ =1.3) for determining the sol-gel state. A: Circular dichroism spectrum changes of (RADA) ₄ peptide, B: Circular dichroism spectrum changes of (RADA) ₃ -RADG peptide.

1. Self-assembling peptides and fusion peptides 1-1. Overview A first aspect of the present invention is a self-assembling peptide and a fusion peptide.
The self-assembling peptide of this embodiment is a peptide consisting of one or more RADA units and one or more RXDA or RADX units arranged in any order, where X represents glycine (G/Gly) or proline (P/Pro). The self-assembling peptide of this embodiment can undergo sol-gel transition at a lower temperature than the known self-assembling peptide (RADA) ₄ peptide.
Furthermore, the fusion peptide of this embodiment is a peptide formed by linking the self-assembling peptide of this embodiment to a functional peptide.

1-2. Definitions of Terms The following terms frequently used in this specification are defined below.
As used herein, the term "self-assembling peptide" refers to a peptide that can be solidified from a sol state dissolved in water or an aqueous solution to a gel state under specific temperature and pressure conditions, such as collagen (including glue, gelatin, and jelly).

"Self-organization" refers to the spontaneous assembly of small molecules in a dispersion medium through intermolecular interactions and the like to form a three-dimensional structure. For example, sugars (polysaccharides such as starch and glucomannan), the aforementioned collagen, and highly absorbent polymers (sodium polyacrylate) self-organize in water or an aqueous solution under specific conditions to form fibrous one-dimensional structures, which then become entangled to form a gel state with a three-dimensional structure. In this specification, substances that gel by self-organization are often referred to as "gelling agents." The self-assembling peptide of the present invention is a type of gelling agent.

A "sol" is a liquid state in which colloidal particles are dispersed in a dispersion medium and have fluidity. For example, a colloid made of a gelling agent is fluidized in a dispersion medium by heating the gel. A "colloid" is a state in which molecules or ions aggregate to form fine particles and are dispersed in a medium. The fine particles that form a colloid are called "colloidal particles."
The "sol state" refers to a liquid state in which colloidal particles are dispersed in a dispersion medium and have fluidity. Generally, this refers to a state in which a gel is fluidized by heating.
The term "solation" refers to a change from a gel state to a sol state.

The term "gel" refers to a substance in which colloidal particles self-organize in a dispersion medium, lose fluidity, and solidify into a solid state.
The term "gel state" refers to a state in which colloidal particles self-organize in a dispersion medium, lose fluidity, and solidify. Generally, this refers to a state in which a sol is solidified by lowering the temperature.
"Gellation" refers to the change from a sol state to a gel state.

"Sol-gel transition" refers to a reversible phase transition phenomenon between sol and gel. In general, the sol-gel transition is temperature dependent under isobaric conditions. In this specification, unless otherwise specified, the sol-gel transition refers to either or both of the transition from sol to gel (gelation) and the transition from gel to sol (solation).

As used herein, the term "fusion peptide" refers to a self-assembling peptide of this embodiment to which a functional peptide is linked via a covalent bond or supramolecular interaction. Covalent bonds include peptide bonds, disulfide bonds, and the like. Supramolecular interactions include hydrogen bonds, hydrophobic interactions, electrostatic interactions, coordinate bonds, and the like. Although not limited thereto, it is preferable that the functional peptide is linked to the N-terminus and/or C-terminus of the self-assembling peptide, and furthermore, the bond is preferably a covalent bond. A more preferred covalent bond is a peptide bond.

In this specification, "gelation temperature" refers to the temperature at which a gelling agent (in this invention, a sol-gel transition agent or a sol-gel transition composition) undergoes a phase transition from a sol state to a gel state. In addition, in this specification, "solation temperature" refers to the temperature at which a gelling agent (in this invention, a sol-gel transition agent or a sol-gel transition composition) undergoes a phase transition from a gel state to a sol state. For a single gelling agent, the gelation temperature and the solation temperature may be the same or different. Generally, whether they are the same temperature or different temperatures usually depends on the type of gelling agent. In this specification, the gelation temperature and the solation temperature are often collectively referred to as the "sol-gel transition temperature."

As used herein, the term "functional peptide" refers to a peptide that has a specific biological function in a living body or within a cell.

In this specification, the term "specific biological function" refers to a function that can affect cells, tissues, or individuals, a function that labels proteins, cells, tissues, and individuals, or a linking function that links other peptides, nucleic acids, low molecular weight compounds, or metal ions that have specific biological functions. "Functions that can affect cells, tissues, or individuals" include, for example, cell adhesion functions, signal transduction functions, binding functions, and metabolic functions. "Specific functions that label proteins, cells, tissues, and individuals" include, for example, fluorescent labels and epitope labels. The specific biological function may be either a natural function or a non-natural function. "Linking functions that link other peptides, nucleic acids, low molecular weight compounds, or metal ions that have specific biological functions" include, for example, functions that mediate antigen-antibody binding or receptor-ligand interactions, functions that bind RNA and/or DNA, and functions that bind nickel ions, copper ions, etc.

As used herein, "biocompatibility" refers to the property of being capable of being introduced into a living organism. In particular, it refers to the property of a material having no toxicity or side effects to a living organism, or the property of the material having only very slight toxicity or side effects, and/or the property of the material not being recognized as a foreign body in the living organism and not being eliminated. As used herein, "a peptide having biocompatibility" refers, for example and without limitation, to a peptide that is free of biological contamination and therefore has no or very little risk of causing allergies or unknown infectious diseases in the human body. Specific examples include chemically synthesized peptides.

As used herein, the term "living body" refers to a cell (including cultured cell), tissue, organ, or individual. Although not limited thereto, the term is preferably a cell of human origin, a tissue or organ composed of cells of human origin, or a human individual.

1-3. Configuration 1-3-1. Self-assembling peptide The configuration of the self-assembling peptide of this embodiment will be specifically explained below.
The self-assembling peptide of this embodiment is composed of a polypeptide in which a plurality of peptide units are linked to each other via peptide bonds.

As used herein, a "peptide unit" refers to a structural unit of a self-assembling peptide in the present invention. One peptide unit is composed of an oligopeptide in which at least three types of amino acids are linked by four peptide bonds.

The peptide units constituting the self-assembling peptide of this embodiment contain three types of amino acids consisting of arginine (Arg/R), alanine (Ala/A), and aspartic acid (Asp/D) as essential constituent amino acids, and may contain other hydrophobic amino acids other than Ala. Hydrophobic amino acids other than Ala include glycine (Gly/G), proline (Pro/P), valine (Val/V), leucine (Leu/L), isoleucine (Ile/I), methionine (Met/M), cysteine (Cis/C), phenylalanine (Phe/F), tyrosine (Tyr/Y), and tryptophan (Trp/W), but any hydrophobic amino acid may be used. Glycine or proline is preferable. The above amino acids other than glycine can be used regardless of their optical isomers. That is, either the D-form or the L-form may be used.

The peptide units constituting the self-assembling peptide of this embodiment are specifically RADA, RXDA, or RADX, where X represents a hydrophobic amino acid other than Ala.

Specifically, the self-assembling peptide of this embodiment is composed of m RADA and n RXDA or RADX linked by peptide bonds. Here, m is an integer between 3 and 6, and n is 1 or 2. Furthermore, there is a relationship between m and n such that 2n<m. Thus, in the self-assembling peptide of this embodiment, the combination of (m, n) is substantially selected from any of (3, 1), (4, 1), (5, 1), (6, 1), (5, 2), or (6, 2).

In the self-assembling peptide of this embodiment, the peptide units are linked in any order. However, in the self-assembling peptide of the present invention, the C-terminal peptide unit is either RXDA or RADX, or the N-terminal peptide unit is RXDA.

Thus, specific examples of the self-assembling peptides of this embodiment include RXDA-(RADA) ₃ (SEQ ID NO:5), (RADA) ₃ -RXDA (SEQ ID NO:6), (RADA) _3- RADX (SEQ ID NO:7), RXDA-(RADA) ₄ (SEQ ID NO:8), (RADA) _4- RXDA (SEQ ID NO:9), (RADA) _4- RADX (SEQ ID NO:10), RXDA-(RADA) ₅ (SEQ ID NO:11), (RADA) _5- RXDA (SEQ ID NO:12), (RADA) _5- RADX (SEQ ID NO:13), RXDA-(RADA) ₆ (SEQ ID NO:14), (RADA) ₆ -RXDA (SEQ ID NO:15), (RADA) _6- RADX (SEQ ID NO:16), (RXDA) _2- (RADA) ₅ (SEQ ID NO:17), RXDA-RADA-RXDA-(RADA) ₄ (SEQ ID NO:18), RXDA-(RADA) ₂ -RXDA-(RADA) ₃ (SEQ ID NO:19), RXDA-(RADA) ₃ -RXDA-(RADA) ₂ (SEQ ID NO:20), RXDA-(RADA) ₄ -RXDA-RADA (SEQ ID NO:21), RXDA-(RADA) ₅ -RXDA (SEQ ID NO:22), RXDA-RADX-(RADA) ₅ (SEQ ID NO:23), RXDA-RADA-RADX-(RADA) ₄ (SEQ ID NO:24), RXDA-(RADA) _2- RADX-(RADA) ₃ (SEQ ID NO:25), RXDA-(RADA) _3- RADX-(RADA) ₂ (SEQ ID NO:26), RXDA-(RADA) _4- RADX-RADA (SEQ ID NO:27), RXDA-(RADA) ₅ -RADX (SEQ ID NO:28), RADA-RXDA-(RADA) _4- RXDA (SEQ ID NO:29), (RADA) ₂ -RXDA-(RADA) ₃ -RXDA (SEQ ID NO:30), (RADA) ₃ -RXDA-(RADA) ₂ -RXDA (SEQ ID NO:31), (RADA) _4- RXDA-RADA-RXDA (SEQ ID NO:32), (RADA) _5- (RXDA) ₂ (SEQ ID NO:33), RADX-(RADA) ₅ -RXDA (SEQ ID NO:34), RADA-RADX-(RADA) _4- RXDA (SEQ ID NO:35), (RADA) _2- RADX-(RADA) _3- RXDA (SEQ ID NO:36), (RADA) ₃ -RADX-(RADA) ₂ -RXDA (SEQ ID NO:37), (RADA) ₄ -RADX-RADA-RXDA (SEQ ID NO:38), (RADA) _5- RADX-RXDA (SEQ ID NO:39), RADA-RXDA-(RADA) _4- RADX (SEQ ID NO:40), (RADA) ₂ -RXDA-(RADA) ₃ -RADX (SEQ ID NO:41), (RADA) ₃ -RXDA-(RADA) ₂ -RADX (SEQ ID NO:42), (RADA) ₄ -RXDA-RADA-RADX (SEQ ID NO:43), (RADA) _5- RXDA-RADX (SEQ ID NO:44), RADX-(RADA) _5- RADX (SEQ ID NO:45), RADA-RADX-(RADA) ₄ -RADX (SEQ ID NO:46), (RADA) ₂ -RADX-(RADA) ₃ -RADX (SEQ ID NO:47), (RADA) ₃ -RADX-(RADA) _2- RADX (SEQ ID NO:48), (RADA) ₄ -RADX-RADA-RADX (SEQ ID NO:49), (RADA) _5- (RADX) ₂ (SEQ ID NO:50), (RXDA) _2- (RADA) ₆ (SEQ ID NO:51), RXDA-RADA-RXDA-(RADA) ₅ (SEQ ID NO:52), RXDA-(RADA) _2- RXDA-(RADA) ₄ (SEQ ID NO:53), RXDA-(RADA) _3- RXDA-(RADA) ₃ (SEQ ID NO:54), RXDA-(RADA) ₄ -RXDA-(RADA) ₂ (SEQ ID NO:55), RXDA-(RADA) ₅ -RXDA-RADA (SEQ ID NO:56), RXDA-(RADA) ₆ -RXDA (SEQ ID NO:57), RXDA-RADX-(RADA) ₆ (SEQ ID NO:58), RXDA-RADA-RADX-(RADA) ₅ (SEQ ID NO:59), RXDA-(RADA) _2- RADX-(RADA) ₄ (SEQ ID NO:60), RXDA-(RADA) _3- RADX-(RADA) ₃ (SEQ ID NO:61), RXDA-(RADA) _4- RADX-(RADA) ₂ (SEQ ID NO:62), RXDA-(RADA) ₅ -RADX-RADA (SEQ ID NO:63), RXDA-(RADA) _6- RADX (SEQ ID NO:64), RADA-RXDA-(RADA) ₅ -RXDA (SEQ ID NO:65), (RADA) ₂ -RXDA-(RADA) _4- RXDA (SEQ ID NO:66), (RADA) _3- RXDA-(RADA) _3- RXDA (SEQ ID NO:67), (RADA) ₄ -RXDA-(RADA) ₂ -RXDA (SEQ ID NO:68), (RADA) ₅ -RXDA-RADA-RXDA (SEQ ID NO:69), (RADA) _6- (RXDA) ₂ (SEQ ID NO:70), RADX-(RADA) ₆ -RXDA (SEQ ID NO:71), RADA-RADX-(RADA) _5- RXDA (SEQ ID NO:72), (RADA) _2- RADX-(RADA) ₄ -RXDA (SEQ ID NO:73), (RADA) ₃ -RADX-(RADA) ₃ -RXDA (SEQ ID NO:74), (RADA) ₄ -RADX-(RADA) ₂ -RXDA (SEQ ID NO:75), (RADA) _5- RADX-RADA-RXDA (SEQ ID NO:76), (RADA) ₆ -RADX-RXDA (SEQ ID NO:77), RADA-RXDA-(RADA) _5- RADX (SEQ ID NO:78), (RADA) ₂ -RXDA-(RADA) ₄ -RADX (SEQ ID NO:79), (RADA) ₃ -RXDA-(RADA) ₃ -RADX (SEQ ID NO:80), (RADA) _4- RXDA-(RADA) ₂ -RADX (SEQ ID NO:81), (RADA) ₅ -RXDA-RADA-RADX (SEQ ID NO:82), (RADA) _6- RXDA-RADX (SEQ ID NO:83), RADX-(RADA) ₆ -RADX (SEQ ID NO:84), RADA-RADX-(RADA) _5- RADX (SEQ ID NO:85), (RADA) _2- RADX-(RADA) _4- RADX (SEQ ID NO:86), (RADA) ₃ -RADX-(RADA) _3- RADX (SEQ ID NO:87), (RADA) _4- RADX-(RADA) ₂ The peptide is any one selected from the group consisting of (RADA) _{5 -RADX-RADA-RADX (SEQ ID NO: 88), (RADA) 5} -RADX-RADA-RADX (SEQ ID NO: 89) and (RADA) ₆ -(RADX) ₂ (SEQ ID NO: 90).

When the self-assembling peptide of this embodiment contains two or more X, the two or more X may be the same amino acid or different amino acids.
The self-assembling peptides of this embodiment can be chemically synthesized.

The self-assembling peptide of this embodiment can control the solation temperature and/or gelation temperature by adjusting the composition and/or arrangement of the peptide units that make up the self-assembling peptide. Specifically, the larger the number m of RADA units, the higher the solation temperature and/or gelation temperature, and the larger the number n of RXDA and RADX, the lower the solation temperature and/or gelation temperature. Therefore, the self-assembling peptide of this embodiment can control its solation temperature and/or gelation temperature by adjusting the values of m and n.

The solization temperature and gelation temperature of the self-assembling peptide of this embodiment are, for example, within a range of 20 to 80°C, within a range of 20 to 70°C, within a range of 20 to 60°C, within a range of 30 to 50°C, or within a range of 30 to 40°C under a condition of 1 atmospheric pressure.
The self-assembling peptide of this embodiment is biocompatible and can be introduced into a living body.

1-3-2. Fusion Peptide The fusion peptide of this embodiment is a peptide obtained by linking a functional peptide to the self-assembling peptide of this embodiment via a covalent bond or supramolecular interaction.

The fusion peptide of this embodiment is, for example and without limitation, a peptide obtained by linking the self-assembling peptide of this embodiment to the N-terminus and/or C-terminus of a functional peptide via a peptide bond. The self-assembling peptide of this embodiment being "linked to the N-terminus and/or C-terminus of a functional peptide via a peptide bond" encompasses the cases where the self-assembling peptide of this embodiment is linked only to the N-terminus of the functional peptide, only to the C-terminus, and both to the N-terminus and C-terminus.

As described above, the functional peptide constituting the fusion peptide of this embodiment is a peptide that has a specific biological function in a living body or a cell. The function may be, but is not limited to, a cell adhesion function, a signal transduction function, a binding function, a labeling function, or a metabolic function.

In addition, the functional peptides constituting the fusion peptide of this embodiment can be selected depending on the purpose of use, the purpose of the fusion peptide, such as cell culture, control of cell adhesion, proliferation, differentiation, etc., tissue or organ culture, formation, regeneration, or vascular induction.

The functional peptides in the present invention include, but are not limited to, cell adhesion molecules, extracellular matrix molecules, secreted proteins, binding proteins, enzymes, marker proteins, and artificial peptides, as well as peptide fragments thereof. The term "cell adhesion molecules" as used herein refers to molecules involved in adhesion between cells or between cells and the extracellular matrix on the surface of cells. Examples of such molecules include, but are not limited to, cadherins such as N-cadherin, integrins, and selectins. The term "extracellular matrix molecules" refers to molecules that constitute the extracellular matrix. Examples of such molecules include, but are not limited to, laminin, collagen, and fibronectin. The term "secreted proteins" refers to proteins that are produced within cells and secreted outside the cells. Examples of such proteins include, but are not limited to, vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), and cytokines. The term "binding proteins" refers to proteins that specifically bind to specific molecules. Examples of functional peptides include, but are not limited to, antibodies or antigens that mediate antigen-antibody binding, (strept)avidin, maltose-binding protein (MBP), receptors or ligands that mediate receptor-ligand interactions, DNA-binding proteins, RNA-binding proteins, and the like. In particular, when the functional peptide is a DNA-binding protein or an RNA-binding protein, the nucleic acid molecule to which the functional peptide binds can bind with one or more other nucleic acid molecules by forming a multiple helix structure, etc. A "marker protein" is a protein that can be used as a label when detecting cells, proteins, etc. Usually, it is a polypeptide that can determine the expression or presence of a target protein based on its activity. Examples of functional peptides include, but are not limited to, fluorescent proteins such as GFP, luminescent proteins such as luciferin or aequorin, and enzymes such as horseradish peroxidase (HRP) or alkaline phosphatase (AP). An "artificial peptide" is also called a tag peptide, and is an oligopeptide consisting of several to a dozen amino acids that is artificially synthesized. Examples of such proteins include epitope tags such as FLAG tags, histidine tags, HA tags, and DAP tags, and His tags.

The solization temperature and gelation temperature of the fusion peptide of this embodiment are influenced by the functional peptide, but are in principle based on the solization temperature and gelation temperature of the self-assembling peptide contained therein. Therefore, under atmospheric pressure, the sol-gel transition occurs, for example, within the range of 20 to 80°C, 20 to 70°C, 20 to 60°C, 30 to 50°C, or 30 to 40°C.

The fusion peptide of this embodiment is biocompatible and can be introduced into a living body.
The self-assembling peptide contained in the fusion peptide of this embodiment has a small molecular weight. Therefore, it is possible to link it to a relatively large functional peptide via a peptide bond and synthesize it as a single peptide chain. In general, when peptide synthesis is performed in cells using a gene expression system, the yield decreases as the molecular weight of the protein increases. The fusion peptide of this embodiment has a high yield when expressed using a gene expression system.

As used herein, the term "gene expression system" refers to an expression system capable of expressing a foreign gene in a host cell, or a cell-free gene expression system. Specific examples include autonomously replicable expression vectors such as plasmids or bacmids. The expression vector may also be a shuttle vector capable of replicating in multiple types of host cells. The host is not particularly limited, but examples include E. coli, insect cells, and cultured cells.

1-4. Effects The self-assembling peptide or fusion peptide of this embodiment has the property of forming a sol at a lower temperature than the (RADA) ₄ peptide.

2. Sol-gel transition agent 2-1. Overview A second aspect of the present invention is a sol-gel transition agent. The sol-gel transition agent of this aspect comprises the self-assembling peptide or fusion peptide of the first aspect. The sol-gel transition agent of this aspect is capable of undergoing sol-gel transition from a sol state to a gel state, or from a gel state to a sol state, by controlling the temperature.

2-2. Configuration In this embodiment, the "sol-gel transition agent" is a gelling agent that is composed of the self-assembling peptide or fusion peptide of the first embodiment and can undergo sol-gel transition at a desired temperature based on the difference in their amino acid sequences.
Therefore, the basic configuration of the sol-gel transition agent in this embodiment is substantially the same as that described in "1-3. Configuration" of the self-assembling peptide of embodiment 1 or the fusion peptide of embodiment 1. Therefore, a detailed description thereof will be omitted here.
The pH of the sol-gel transition agent in this embodiment is not limited, and may be, for example, in the range of pH 1 to pH 11, pH 1 to pH 9, pH 3 to pH 9, pH 4 to pH 8, pH 5 to pH 7, or pH 6 to pH 7. The pH is preferably 8.0 or less, pH 7.5 or less, pH 7 or less, or pH 6.5 or less.

3. Sol-gel transition composition 3-1. Overview The third aspect of the present invention is a sol-gel transition composition. The sol-gel transition composition of this aspect contains the sol-gel transition agent described in the second aspect as an essential active ingredient, and also contains a carrier and the like. The sol-gel transition composition of this aspect can cause the sol-gel transition agent to undergo sol-gel transition at a desired temperature by temperature treatment.
As used herein, the term "temperature treatment" refers to a treatment for changing the temperature, such as a treatment for increasing the temperature (heating treatment) or decreasing the temperature (cooling treatment) of the sol-gel transition agent or sol-gel transition composition of the present invention in a sol or gel state.
As used herein, the term "causing a sol-gel transition due to a temperature change" refers to causing a sol-gel transition of a sol-gel transition agent or a sol-gel transition composition by temperature treatment under any pressure condition, for example, causing a sol-gel transition due to a temperature change within a range of 20 to 80° C. under 1 atmospheric pressure.

3-2. Composition 3-2-1. Constituent Components The sol-gel transition composition of the present invention is composed of an active ingredient and other components. The components other than the active ingredient are not particularly limited, and examples thereof include a carrier. Each of the constituent components will be described in detail below.

(1) Active Ingredient The sol-gel transition composition of this embodiment contains one or more sol-gel transition agents according to the second embodiment as an essential active ingredient, i.e., the self-assembling peptide and/or fusion peptide according to the first embodiment. The sol-gel transition agent according to the second embodiment as an active ingredient may be one type or a combination of two or more different types.

The sol-gel transition composition of this embodiment may further include, as another active ingredient, a self-assembling peptide consisting of (RADA) _p (p is an integer of 1 to 6), and/or a fusion peptide formed by linking a functional peptide to the self-assembling peptide via a covalent bond or supramolecular interaction. These self-assembling peptides have a higher sol-gel transition temperature than the sol-gel transition agent of the present invention, and remain in a gel state at, for example, 20 to 80°C. Therefore, by mixing these self-assembling peptides with the sol-gel transition agent of the present invention, the sol-gel transition temperature in the sol-gel transition composition can be upregulated, that is, set to a temperature higher than that of the sol-gel transition agent alone. This makes it possible to control the temperature of the sol-gel transition in the sol-gel transition composition. Furthermore, the sol-gel transition composition of this embodiment may also include a drug or the like as another active ingredient. In this specification, the term "drug" is a concept that includes low molecular weight compounds, peptides (including enzymes and antibodies), or nucleic acids (including RNAi molecules such as miRNA, siRNA, and shRNA, antisense nucleic acids, aptamers, etc.). Drugs include, but are not limited to, various types of drugs, such as therapeutic drugs intended to treat diseases or alleviate symptoms, diagnostic drugs for detecting or diagnosing diseases, agricultural chemicals intended to repel or exterminate pests and vermin, disinfectants intended to kill viruses or bacteria, etc. The drug contained in the sol-gel transition composition of this embodiment may be one type or two or more types.

The amount (content) of the sol-gel transition agent described in the second aspect to be mixed in the sol-gel transition composition is not particularly limited. It may be appropriately determined taking into account the conditions of the sol-gel transition temperature. In addition, when the sol-gel transition composition of the present invention is administered to a living body, it may be appropriately determined according to the type and/or effective amount of the sol-gel transition agent contained in the sol-gel transition composition, information on the subject, the dosage form of the sol-gel transition composition, and the type of carrier or additive described later. Specifically, the concentration of the sol-gel transition agent described in the second aspect in the sol-gel transition composition is not limited, but may be, for example, 0.4% by weight to 10% by weight, 1.0% by weight to 10% by weight, or 1.5% by weight to 10% by weight. In this specification, the "effective amount" refers to an amount necessary for the sol-gel transition agent to function as an active ingredient in the sol-gel transition composition and an amount that gives little or no harmful side effects to the living body to which it is applied. This effective amount may vary depending on various conditions such as information on the subject, the route of administration, and the number of administrations. Here, the term "subject" refers to a living body to which the pharmaceutical composition is applied. For example, this includes humans, livestock (cattle, horses, sheep, goats, pigs, chickens, ostriches, etc.), racehorses, pets (dogs, cats, rabbits, etc.), and experimental animals (mice, rats, guinea pigs, monkeys, marmosets, etc.). Preferably, the subject is a human (in this case, it is particularly referred to as a "subject"). In addition, "subject information" refers to various individual information of the living body to which the sol-gel transition composition is applied, and in the case of a subject, for example, the subject's overall health condition, the progression and severity of a disease or injury if the subject is suffering from such a disease, age, weight, sex, diet, drug sensitivity, the presence or absence of concomitant drugs, and resistance to treatment. The final effective amount of the sol-gel transition agent and the application amount calculated based on this are ultimately determined by the judgment of a doctor, dentist, veterinarian, etc., depending on the information of each individual subject.

(2) Carrier The sol-gel transition composition of this embodiment can contain a pharma- ceutically acceptable carrier, if necessary.
As used herein, the term "pharmaceutical acceptable carrier" refers to additives commonly used in the field of pharmaceutical formulations, such as solvents, excipients, fillers, emulsifiers, flow regulators, lubricants, human serum albumin, and the like.

The solvent may be, for example, water or any other pharma- ceutically acceptable aqueous solution, or a pharma-ceutically acceptable organic solvent. Examples of aqueous solutions include physiological saline, isotonic solutions containing glucose or other adjuvants, phosphate buffer, and sodium acetate buffer. Examples of adjuvants include D-sorbitol, D-mannose, D-mannitol, sodium chloride, low-concentration nonionic surfactants, polyoxyethylene sorbitan fatty acid esters, and the like.

Excipients may include, for example, sugars such as monosaccharides, disaccharides, cyclodextrins and polysaccharides, metal salts, citric acid, tartaric acid, glycine, polyethylene glycol, pluronics, kaolin, silicic acid, or combinations thereof.

Examples of fillers include petrolatum, sugars and/or calcium phosphate.
Examples of emulsifiers include sorbitan fatty acid esters, glycerin fatty acid esters, sucrose fatty acid esters, and propylene glycol fatty acid esters.

Examples of flow regulators and lubricants include silicates, talc, stearates or polyethylene glycol.

In addition to the above, if necessary, the composition may contain appropriate solubilizers, suspending agents, diluents, dispersing agents, surfactants, soothing agents, stabilizers, absorption promoters, bulking agents, moisturizing agents, humectants, adsorbents, flavoring agents, disintegration inhibitors, coating agents, colorants, preservatives, antiseptics, antioxidants, fragrances, flavorings, sweeteners, buffers, isotonicity agents, etc. that are commonly used in pharmaceuticals.

Such carriers are primarily used to facilitate the formation of dosage forms and maintain the dosage form and drug efficacy, as well as to make the active ingredient, the sol-gel transition agent, less susceptible to decomposition by enzymes in the body, etc., and may be used appropriately as needed.

3-2-2. Properties of the sol-gel transition composition The pH of the sol-gel transition composition of this embodiment is not limited. For example, it may be in the range of pH 1 to pH 11, pH 1 to pH 9, pH 3 to pH 9, pH 4 to pH 8, pH 5 to pH 7, or pH 6 to pH 7. The pH is preferably 8.0 or less, pH 7.5 or less, pH 7 or less, or pH 6.5 or less.

The sol-gel transition composition of this embodiment can also undergo sol-gel transition by temperature treatment under a predetermined pressure condition. The sol-gel transition temperature is also based on that of the self-assembling peptide or fusion peptide constituting the sol-gel transition agent, which is the active ingredient, in principle. Therefore, the sol-gel transition composition of this embodiment can undergo sol-gel transition at a temperature within the range of 20 to 80°C, 20 to 70°C, 20 to 60°C, 30 to 50°C, or 30 to 40°C under 1 atmosphere, for example. The sol-gel transition composition of this embodiment undergoes sol-gel transition at a temperature higher than the body temperature of the subject under 1 atmosphere, for example. Therefore, the sol-gel transition composition of this embodiment can be adjusted based on the sol-gel transition temperature of the sol-gel transition agent, including the sol-gel transition temperature. For example, if you want to cause the sol-gel transition composition of this embodiment to undergo sol-gel transition at a temperature higher than the normal body temperature of the subject (e.g., 37°C) under 1 atmosphere, if there is only one type of sol-gel transition agent, which is the active ingredient, you can select a sol-gel transition agent that has a sol-gel transition temperature higher than the normal body temperature. If there are two types of sol-gel transition agents as active ingredients, the result can be obtained by combining a sol-gel transition agent having a sol-gel transition temperature clearly higher than normal body temperature (e.g., 40°C or higher, 45°C or higher, 47°C or higher, or 50°C or higher) with a sol-gel transition agent having a sol-gel transition temperature clearly lower than normal body temperature (e.g., 35°C or lower, 32°C or lower, 30°C or lower, or 28°C or lower).

Therefore, in one embodiment of the sol-gel transition composition of this aspect, it can be administered to a subject in a sol state and allowed to gel in vivo, or conversely, it can be administered to the body in a gel state and then allowed to solize at a desired time.

When the sol-gel transition composition of this embodiment contains a fusion peptide, after the sol-gel transition composition of this embodiment gels in the body of a subject, a function based on the biological function of the functional peptide constituting the fusion peptide is exerted. For example, when VEGF is used as the functional peptide, the sol-gel transition composition that gels in the body can induce angiogenesis in the gel. Furthermore, after angiogenesis has been sufficiently induced by the sol-gel transition composition in a gel state at the administration site, the sol-gel transition composition can be resolized by temperature treatment and removed from the administration site.

The sol-gel transition composition of this embodiment can also be used as a cell scaffold in vivo. In this case, after the sol-gel transition composition of this embodiment gels in the body of the subject, the function based on the biological function of the functional peptide constituting the fusion peptide is exerted. For example, extracellular matrix molecules such as laminin and N-cadherin can be used as the functional peptide. When used as a cell scaffold, the sol-gel transition composition that has gelled in the body enables the control of cell adhesion, proliferation, differentiation, etc., and the formation, regeneration, etc. of tissues or organs in the gel. Furthermore, after the control of cell adhesion, proliferation, differentiation, etc., and the formation, regeneration, etc. of tissues or organs are sufficiently achieved by the sol-gel transition composition in a gel state at the administration site, the sol-gel transition composition can be resolized by temperature treatment and removed from the administration site.

When the sol-gel transition composition of this embodiment contains a fusion peptide or a drug, the effect may be controlled so that it is not exerted in the gel state but is exerted after solization. For example, the sol-gel transition composition of this embodiment is administered in a sol state to an internal site different from the action site of the functional peptide, gelled at the administration site, and then maintained in a gel state until a required time. Thereafter, the administration site is subjected to a temperature treatment at a required time to solize the composition, release the fused peptide contained therein, and deliver the fusion peptide to the action site of the functional peptide contained in the fusion peptide.

The sol-gel transition composition of this embodiment can also be introduced to a subject in a gel state. For example, the step of administering the composition in a sol state and gelling it inside the subject's body can be omitted from the above-mentioned usage mode, and the composition can be transplanted into the living body in a gel state by a surgical method or the like instead.

The sol-gel transition composition of this embodiment introduced into a subject can also be removed from the introduction site without being resol-ified by temperature treatment. For example, it can be surgically removed from the introduction site while still in a gel state from the body of the subject.

3-2-3. Dosage Form The dosage form of the sol-gel transition composition of this embodiment is not particularly limited. Any form may be used as long as it can be delivered to the target site without inactivating the properties of the active ingredient in the body of the subject. For example, a liquid that can be directly administered to the target site may be mentioned. An example of a liquid is an injection. An injection can be formulated by appropriately combining the above-mentioned excipients, stabilizers, pH adjusters, etc. Injections are generally liquids, and therefore the sol-gel transition composition as an injection is in a sol state.
On the other hand, the dosage form of the sol-gel transition composition of this embodiment may be a solid formulation that can be introduced into a target site. In the case of a solid formulation, the shape is not important. In addition to general solid formulation forms such as powders, powders, granules, and tablets, the form may be that of a transplant member. Since a solid formulation is a solid, the sol-gel transition composition as a solid formulation is usually in a gel state.

3-2-4. Application Method The application method of the sol-gel transition composition of this embodiment is not particularly limited, but is preferably parenteral administration, and more preferably local administration. Local administration includes, for example, intramuscular administration, subcutaneous administration, tissue administration, and organ administration. When the sol-gel transition composition of this embodiment is administered locally, the sol-gel transition composition of this embodiment in a sol state is directly administered to the target site by injection or the like, and can gel at the target site after administration. Alternatively, the sol-gel transition composition of this embodiment may be introduced to the target site in a gel state. For example, the target site can be incised by surgical operation, and the composition can be transplanted in a gel state. The dosage may be an amount effective for the active ingredient to be effective. The effective amount is appropriately selected according to the subject information.

3-2-5. Removal Method After the sol-gel transition composition of this embodiment has gelled at the administration site, it can be resolvated by temperature treatment as necessary and then removed from the administration site.
Alternatively, the sol-gel transition composition of this embodiment may be removed from the target site while still in the gel state. For example, the administration site can be incised by surgical operation and the composition can be surgically removed while still in the gel state.
The timing for converting the sol-gel transition composition of this embodiment into a sol and removing it from the administration site can be appropriately determined as necessary.

4. Cell scaffold 4-1. Overview The fourth aspect of the present invention is a cell scaffold. The cell scaffold of this aspect is composed of the sol-gel transition agent described in the second aspect or the sol-gel transition composition described in the third aspect. The cell scaffold of this aspect can be used either in vitro or in vivo. The shape of the cell scaffold of this aspect can be processed by temperature treatment.

4-2. Configuration In this specification, the term "cell scaffold" refers to a scaffold material for cell culture or tissue regeneration, which is a material that enables the control of cell adhesion, proliferation, differentiation, etc., and the culture, formation, regeneration, etc. of transplanted cells, transplanted tissues, or transplanted organs. The cell scaffold material may be used in vitro or in vivo.

As used herein, "control of cell adhesion, proliferation, differentiation, etc." refers to the promotion and/or inhibition of cell adhesion, proliferation, and differentiation.

The cell scaffold material of this embodiment is composed of the sol-gel transition agent described in the second embodiment or the sol-gel transition composition described in the third embodiment. Therefore, the basic configuration of this embodiment is substantially the same as the configuration of the sol-gel transition agent of the second embodiment or the sol-gel transition composition described in the third embodiment.

The cell scaffold material of this embodiment is, in principle, a sol-gel transition agent in a gel state. This is because, in order to function as a cell scaffold, a certain degree of rigidity is required for the cells to adhere, and this purpose cannot usually be achieved with a liquid sol.

When the cell scaffold material of this embodiment contains a fusion peptide, the functional peptide constituting the fusion peptide is, for example, a molecule that controls cell adhesion, proliferation, differentiation, etc., or a molecule that controls tissue or organ formation, regeneration, etc. Examples are cell adhesion molecules, extracellular matrix molecules, secreted proteins, binding proteins, enzymes, marker proteins, and artificial peptides, as well as peptide fragments thereof. The functional peptides constituting the fusion peptide that can be included in the cell scaffold material of this embodiment are preferably cell adhesion molecules and extracellular matrix molecules, such as laminin and N-cadherin.

4-3. Effects The cell scaffold of this embodiment enables control of cell adhesion, proliferation, differentiation, etc., in vitro or in vivo, or enables formation, regeneration, etc., of tissues or organs.
The mode of use of the cell scaffold material of this embodiment in vivo is described in "3-2. Constitution" of "3. Sol-gel transition composition," and a detailed description thereof will be omitted here.
When using the cell scaffold of this embodiment, the shape of the cell scaffold can be processed to match the desired shape of the target cell, tissue, or organ. For example, the cell scaffold of the present invention can be molded by pouring it into a mold in a sol state and gelling it. Alternatively, the cell scaffold of the present invention can be molded into the desired shape by locally heating the gel to turn it into a sol.

5. Sol-gel transition method 5-1. Overview A fifth aspect of the present invention is a method for performing a sol-gel transition of the sol-gel transition agent or sol-gel transition composition of the present invention, which is in a sol state or a gel state.
The sol-gel transition method of the present invention includes a temperature treatment step as an essential step.

5-2. Method 5-2-1. Temperature treatment step The "temperature treatment step" refers to a step of increasing the temperature from a temperature lower than the sol-forming temperature of the sol-gel transition agent or sol-gel transition composition of the present invention to the said temperature or higher, or decreasing the temperature from a temperature higher than the gelling temperature to the said temperature or lower. This step enables the sol-gel transition agent or sol-gel transition composition of the present invention to undergo a phase transition from a sol state to a gel state, or from a gel state to a sol state.

The treatment temperature in this process varies depending on the type of sol-gel transition agent or sol-gel transition composition used, and may be appropriately determined depending on the type. For example, the temperature may be increased or decreased within the range of 10 to 90°C, 20 to 80°C, 20 to 70°C, 20 to 60°C, 30 to 50°C, or 30 to 40°C.

The heating method or cooling method used in the temperature treatment step in the sol-gel transition method of the present invention is not particularly limited. Any known method may be used. For example, the heating method may include a method of directly or indirectly contacting the sol-gel transition agent or the sol-gel transition composition with a heat source (direct flame, hot water bath, heater irradiation, etc.) or a method of irradiating with microwaves or ultrasonic waves. In addition, the cooling method may include a method of placing the sol-gel transition agent or the sol-gel transition composition in a freezer or refrigerator.
This process can be carried out either in vitro or in vivo.

Example 1: Identification of a self-assembling peptide that undergoes sol-gel transition in the temperature range of 20 to 80°C
(the purpose)
We will develop self-assembling peptides that undergo sol-gel transition in the temperature range of 20 to 80°C.
(Method)
(1) Synthesis of self-assembling peptides A total of eight peptides: (RADA) ₄ peptide, RGDA-(RADA) ₃ peptide, RADG-(RADA) ₃ peptide, (RADA) ₂ -RGDA-RADA peptide, (RADA) ₂ -RADG-RADA peptide, (RADA) ₃ -RGDA peptide, (RADA) ₃ -RADG peptide, and (RADA) ₄ -G peptide, were synthesized on a 0.10 mmol scale by Fmoc peptide solid-phase synthesis using polystyrene resin as follows.

In a solid-phase synthesis tube (Hi-Pep Laboratory Co., Ltd., solid-phase synthesis tube polypropylene LibraTube main tube 5 mL, solid-phase synthesis cap polypropylene LibraTube upper cap), Fmoc-NH-SA resin (Watanabe Chemical Co., Ltd.) (250 mg, 0.10 mmol) was immersed in N,N'-dimethylformamide (DMF) (Kishida Chemical Co., Ltd.) overnight and allowed to swell. Piperidine (Kishida Chemical Co., Ltd.) (20% in DMF, 2 mL) was added, the mixture was stirred with a vortex for 1 minute, and the reaction solution was then removed. Piperidine (20% in DMF, 2 mL) was added, the mixture was shaken at room temperature for 10 minutes, and the reaction solution was then removed. The mixture was washed five times with DMF (2 mL) to remove the solvent. A small amount of the resin was taken out, and the resin was confirmed to be colored using a TNBS Test Kit (Tokyo Chemical Industry Co., Ltd.). The resin was washed three times each with methylene chloride (Godoh Co., Ltd.) (2 mL) and DMF (2 mL), and the solvent was then removed. The N-terminal amino acid (0.30 mmol) was dissolved in a condensation agent cocktail (700 μL), a mixture of N,N-diisopropylethylamine (DIEA) (Nacalai Tesque, Inc.) and N-methyl-2-pyrrolidone (NMP) (Kishida Chemical Co., Ltd.) (DIEA/NMP = 2.75/14.25 (v/v), 700 μL), and added to the resin. The condensation agent cocktail was prepared by mixing 3.05 g of HBTU (Watanabe Chemical Co., Ltd.), 1.25 g of HOBt·H ₂ O (Watanabe Chemical Co., Ltd.), and 16 mL of DMF. The mixture was shaken at room temperature for 15 minutes, after which the reaction solution was removed. The mixture was washed five times with DMF (2 mL) to remove the solvent. A small amount of the resin was taken out, and it was confirmed that the resin did not color using the TNBS Test Kit (Tokyo Chemical Industry Co., Ltd.). The mixture was washed three times each with methylene chloride (2 mL) and DMF (2 mL), and the solvent was removed. The above procedure was then repeated according to the amino acid sequence to elongate the peptide chain. Fmoc-Ala- _OH.H2O , Fmoc-Asp(OtBu)-OH, Fmoc-Arg(Pbf)-OH and Fmoc-Gly-OH (Watanabe Chemical Co., Ltd.) were used for peptide chain elongation.

After peptide elongation, the solvent was removed, acetic anhydride (Kanto Chemical Co., Ltd.) (25% methylene chloride solution, 2 mL) was added, and the mixture was shaken at room temperature for 5 minutes, after which the reaction solution was removed. The mixture was washed five times with DMF (2 mL) and the solvent was removed. A small amount of the resin was removed and confirmed to be non-colored using the TNBS Test Kit. The mixture was washed three times each with methylene chloride (2 mL) and DMF (2 mL), and the solvent was removed.

Next, the peptide was cleaved from the resin and lyophilized using the following procedure. The deprotection cocktail was added to the resin dried in a desiccator, and the resin was gently shaken every 30 minutes at room temperature and allowed to stand for 90 minutes. The deprotection cocktail was prepared by mixing 2375 μL of trifluoroacetic acid (TFA) (Kishida Chemical Co., Ltd.), 62.5 μL of triisopropylsilane (TIS) (Tokyo Chemical Industry Co., Ltd.), and 62.5 μL of water in advance. The filtrate was collected in a 15 mL centrifuge tube. TFA (500 μL) was added to the synthesis tube, and the filtrate was collected in the same centrifuge tube. This operation was repeated three times. Diethyl ether (Kishida Chemical Co., Ltd.) (40 mL) was added to the centrifuge tube in which the filtrate was collected, and the mixture was thoroughly stirred. The mixture was centrifuged (4°C, 3500×g, 5 minutes) and the supernatant was removed. This operation was repeated three times. The mixture was left to stand in a fume hood for 10 minutes, dried, and then dried in a desiccator for more than two hours. The dried sample was dispersed in ion-exchanged water and freeze-dried.

(2) Preparation of gels containing self-assembling peptides
In a 2 mL glass screw vial (AS ONE, Labolan screw vial), 2.2% trifluoroacetic acid aqueous solution (200 μL) was added to each peptide powder (1.0 mg) lyophilized in (1), and the sample was solated by ultrasonic irradiation at 25°C for 5 minutes using a water bath type ultrasonicator (AS12GTU, 35 kHz, 60 W). The peptide concentration was 0.50 wt%. The vial was then stood vertically and left overnight in a thermostatic bath (Mitsubishi Electric Engineering, Cool Incubator CN-40A) at 20°C to gel the self-assembling peptides. Two or more vials were prepared for each peptide. As shown in Figure 1, the distance from the bottom of the container to the top of the gel sample was measured as the reference distance h _0. As a result, h ₀ = 6 mm for all peptide samples.

(3) Sol-gel transition assay For each peptide sample, the vial prepared in (2) was tilted 90 degrees, i.e., horizontally, at 20°C. The distance from the bottom of the container to the end of the sample moved by solization after 1 minute was measured as the movement distance h. Next, for each peptide sample, another vial prepared in (2) was heated to 80°C (temperature change rate = 5°C/min), incubated at 80°C for 5 minutes, and then the movement distance when tilted horizontally was measured as at 20°C. The state of each peptide sample at each temperature was judged. The h/h ₀ value was used as the judgment criterion, and h/h ₀ = 1.3 was used as the cutoff value for the sol-gel state. That is, if the h/h ₀ value of the peptide sample was 1.3 or more, it was in the sol state, and if it was less than 1.3, it was in the gel state.

(result)
Figure 2 shows the h/h ₀ value of each peptide sample at 20° C. (RADA) ₄ peptide, RGDA-(RADA) ₃ peptide, (RADA) ₃ -RGDA peptide, (RADA) ₃ -RADG peptide, and (RADA) ₄ -G peptide were in a gel state, while the other peptides were in a sol state.
3 shows the h/h ₀ of each peptide sample at 80° C. The (RADA) ₄ peptide and (RADA) ₄ -G peptide were determined to be in a gel state, while the others were determined to be in a sol state.
From the results at 20°C and 80°C, the RGDA-(RADA) ₃ peptide, the (RADA) ₃ -RGDA peptide, and the (RADA) ₃ -RADG peptide showed a phase transition from gel to sol between 20°C and 80°C. On the other hand, the (RADA) ₄ peptide and the (RADA) ₄ -G peptide remained in a gel form at both 20°C and 80°C. The RADG-(RADA) ₃ peptide, the (RADA) ₂ -RGDA-RADA peptide, and the (RADA) ₂ -RADG-RADA peptide were in a sol form at both 20°C and 80°C.

Example 2: Measurement of circular dichroism spectra of self-assembling peptides in the temperature range of 20 to 80°C
(the purpose)
The conformational changes of the self-assembling peptides are verified by measuring circular dichroism spectra.
The term "circular dichroism spectrum change" refers to a change in the circular dichroism spectrum associated with a change in the three-dimensional structure of a protein. "Circular dichroism" refers to a phenomenon in which a difference in absorbance occurs between left-handed and right-handed circularly polarized light when a protein absorbs circularly polarized light. When the three-dimensional structure of a protein changes, a change in the circular dichroism spectrum occurs. For example, when the self-assembling peptide of the present invention undergoes a sol-gel transition, a change in the circular dichroism spectrum is observed, and when the self-assembling peptide of the present invention undergoes a transition from gel to sol, a change in the circular dichroism spectrum that is thought to be due to the collapse of the β-sheet structure is observed. In this example, the purpose is to evaluate the change in the formation ratio of the β-sheet structure required for gel formation of the self-assembling peptide by measuring the change in the circular dichroism spectrum.

(Method)
Circular dichroism spectra were measured for (RADA) ₄ peptide and (RADA) ₃ -RADG peptide at a concentration of 0.50 wt% in 2.2% TFA aqueous solution when the temperature was raised from 20° C. to 40° C., 60° C., and 80° C. Furthermore, the circular dichroism spectra of the self-assembling peptides were measured when the temperature was lowered again to 20° C.
The peptide samples were synthesized in the same manner as in Example 1.
Circular dichroism spectra were measured using a JASCO J-1100 CD Spectrometer with an assembled cell (GL Science, AB20-UV-0.1, cell length 0.1 mm). The measurement range was 190-400 nm, the data acquisition interval was 0.2 nm, the scan speed was 200 nm/min, and the sample concentration was 0.50 wt%. Temperature control during the measurement of circular dichroism spectra was performed using a JASCO temperature control unit and a water-cooled Peltier cell holder PTC-514.

(result)
The (RADA) ₄ peptide did not show any spectral change when heated from 20 to 80 °C (Fig. 4A). In other words, the intensity of the negative Cotton effect at around 220 nm, which is derived from the β-sheet structure, did not decrease with increasing temperature, indicating that the β-sheet structure did not collapse. This is consistent with the fact that the (RADA) ₄ peptide did not form a sol even when heated.
On the other hand, in the case of the (RADA) ₃ -RADG peptide, the intensity of the negative Cotton effect around 220 nm, which is derived from the β-sheet structure, decreased with increasing temperature, indicating the collapse of the β-sheet structure (Fig. 4B). This was consistent with the (RADA) ₃ -RADG peptide converting to a sol when heated. Furthermore, when the temperature was lowered again to 20°C, the intensity of the negative Cotton effect around 220 nm recovered, indicating the reformation of the β-sheet structure. Thus, it was shown that the sol-gel transition in the (RADA) ₃ -RADG peptide is reversible.
All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims (12)

Any self-assembling peptide selected from the group consisting of amino acid sequences represented by RGDA-(RADA) ₃ , (RADA) ₃ -RGDA, and (RADA) ₃ -RADG. A fusion peptide comprising the self-assembling peptide according to claim 1 and a functional peptide linked thereto via a covalent bond or supramolecular interaction. The fusion peptide according to claim 2, wherein the functional peptide is any one of functional peptides selected from laminin, VEGF, and N-cadherin. A sol-gel transition agent comprising the self-assembling peptide of claim 1 or the fusion peptide of claim 2 or 3. A sol-gel transition composition comprising the sol-gel transition agent according to claim 4 as an active ingredient. The sol-gel transition composition according to claim 5, comprising two or more sol-gel transition agents according to claim 4 . ( RADA ) _p (p is an integer from 1 to 6), and /or
The sol-gel transition composition according to claim 5 or 6, further comprising a fusion peptide comprising a self-assembling peptide consisting of (RADA) _p (p is an integer from 1 to 6) and a functional peptide linked to the self-assembling peptide via a covalent bond or supramolecular interaction. A cell scaffold material comprising the sol-gel transition agent according to claim 4 in a gel state, and/or the sol-gel transition composition according to any one of claims 5 to 7. A method for sol-gel transition of the sol-gel transition agent according to claim 4 or the sol-gel transition composition according to any one of claims 5 to 7, which is in a sol or gel state, comprising the steps of:
The method further comprises a temperature treatment step of treating the sol-gel transition agent or the sol-gel transition composition at a temperature equal to or higher than its solization temperature or equal to or lower than its gelation temperature. The method according to claim 9, wherein the concentration of the sol-gel transition agent and/or the sol-gel transition composition is 0.4% by weight or more and 10% by weight or less. The method according to claim 9 or 10, wherein the sol-gel transition agent or the sol-gel transition composition in a sol or gel state has a pH of 1 to 9. The method according to any one of claims 9 to 11, wherein the solization temperature or gelation temperature is within the range of 20 to 80°C.

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